The present invention relates to a mass spectrometer having an ion accumulator and a time-of-flight mass spectrometer coupled thereto and more particularly, to the provision of a mass spectrometer having both the function of multi-stage tandem mass spectrometry (MSn) and a high mass accuracy of less than 5 ppm.
With advanced genome decryption for a background, a proteome analysis for comprehensively analyzing protein appearing in vivo has been noticed. An analyzing method using the mass spectrometer features high sensitivity and high throughput and serves as a leading technique for the proteome analysis. Of the analyzing methods, the tandem mass spectrometry (MSn) technique can improve the analytical efficiency of the proteome analysis drastically and therefore has been thought much of.
As a mass spectrometer capable of performing the MSn spectrometry, an ion trap mass spectrometer described in U.S. Pat. No. 2,939,952 is known. In the ion trap mass spectrometer, a quadrupole electric field is formed inside an ion trap by applying an RF voltage so as to capture and accumulate ions and subsequently, accumulated ions are ejected and detected in order of smaller mass-to-charge ratios of ions by scanning the amplitude of the RF voltage, thereby undergoing mass spectrometry. In the ion trap mass spectrometer, an MS2 spectrometry is made as follows. Firstly, ions are accumulated in the ion trap. Next, ions in an arbitrarily selected mass range are kept to remain and ions in other mass ranges are ejected out of the ion trap (this operation is called xe2x80x9cisolationxe2x80x9d). Thereafter, the selected ions (parent ions) are dissociated to create fragment ions (daughter ions) which in turn are captured in the ion trap. Finally the RF voltage is scanned, with the result that accumulated daughter ions are ejected and detected in order of smaller mass-to-charge ratios (m/z) of the daughter ions so as to undergo mass spectrometry. Out of the daughter ions, ions in a specified mass range are selected and the selected ions now personate parent ions to be applied with an operation similar to the above to create daughter ions which in turn undergo mass spectrometry. This is an MS3 spectrometry. By repeating a similar operation, an MSn spectrometry can proceed. The dissociation of parent ions is achieved through collision induced dissociation (CID). In the CID, a neutral gas (target gas) is introduced into the ion trap and is caused to collide with ions to dissociate them. The MSn spectrometry can give detailed information of the structure of an analyte and is therefore a technique effective for analysis of a structure of an unknown substance. The ion trap mass spectrometer, however, faces a problem that the mass accuracy is poor because of space charge effects. The space charge effects referred to herein signify that the quadrupole electric field for capturing ions is affected by perturbation due to charges of captured ions. The larger the amount of captured ions, the more the space charge effects become noticeable, so that ions are lost and the mass resolution of mass spectrum and mass accuracy are degraded.
Known reference 1 (U.S. Pat. No. 5,572,022) discloses a method and apparatus for operating an ion trap mass spectrometer in such a manner that space charge effects do not become noticeable. A dissolved sample delivered out of a liquid chromatograph, for instance, is ionized in an ion source and then admitted to an ion trap. By controlling a lens system arranged immediately before the ion trap, ions can be admitted to the ion trap for a constant period of time. In the case of an MSn spectrometry, selection and dissociation of parent ions are performed. Finally, by scanning an RF voltage, mass spectrometry of ions captured in the ion trap is carried out. This operation is repeated until delivery of the dissolved sample from the liquid chromatograph ends. At that time, the time for introducing ions into the ion trap is determined on the basis of total content of ions detected in a mass spectrometry carried out immediately precedently and a threshold value preset in advance. The threshold value referred to herein is set to an amount of ions with which the space charge effects will not become noticeable. In the ion trap mass spectrometer, however, a neutral gas prevails inside the ion trap and hence collisions of ions with the gas take place even during mass spectrometry. Since a cross-section of collision with the gas differs to a great extent depending on the kind of ions and even for ions of the same m/z (mass-to-charge ratio), the ions are detected at shifted times. This makes it difficult to attain a mass accuracy of less than 0.1 amu (atomic mass unit) with the ion trap mass spectrometer.
Known reference 2 (B. M. Chien, S. M. Michael and D. M. Lubman, Rapid Commun. Mass in Spectrometry, Vol. 7, 837. (1993)) discloses an apparatus having an ion trap and a time-of-flight mass spectrometer coupled thereto. In the apparatus, a process up to capture and isolation of ions and dissociation of ions is carried out inside the ion trap and mass spectrometry of daughter ions is performed by means of the time-of-flight mass spectrometer. The time-of-flight mass spectrometer features a high mass accuracy of less than 5 ppm. But in the apparatus, the ion trap also serves as a portion of the time-of-flight mass spectrometer (accelerator) and as a result, collisions of ions with a neutral gas take place during mass spectrometry. Consequently, measurement accuracy of time of flight, accordingly, mass resolution and mass accuracy are degraded.
Known reference 3 (JP-A-2001-297730) discloses another type of apparatus having an ion trap and a time-of-flight mass spectrometer in combination. In the apparatus, a process up to capture and isolation of ions and dissociation of ions are carried out inside the ion trap and mass spectrometry of daughter ions is performed by means of the time-of-flight mass spectrometer. In the apparatus, the ion trap and the mass spectrometer are separated from each other, so that ions accumulated in the ion trap are once ejected out of the ion trap and then introduced to the time-of-flight mass spectrometer so as to undergo mass spectrometry therein. Inside the time-of-flight mass spectrometer, an acceleration field is formed in a direction orthogonal to the traveling direction of ions and time of flight required for ions to reach a detector from an accelerator is measured. The interior of the time-of-flight mass spectrometer is maintained at high vacuum and collisions of ions with gas hardly take place therein. Therefore, an MSn spectrometry can be executed at a high mass accuracy the time-of-flight mass spectrometer has.
Disadvantageously, in the ion trap mass spectrometer, loss of ions occurs inside the ion trap owing to the space charge effects and the mass resolution of mass spectrum and mass accuracy are degraded. In the known reference 1, the amount of ions accumulated in the ion trap can be so adjusted as to prevent the space charge effects from becoming noticeable but mass spectrometry is performed by means of the ion trap and there results a mass accuracy of only less than 0.1 amu. This accuracy is insufficient for proteome analysis. In the known reference 2, the apparatus is disclosed in which mass spectrometry of ions accumulated in the ion trap is performed by means of the time-of-flight mass spectrometer having high mass accuracies. But, since the ion trap also serves as an accelerator of the time-of-flight mass spectrometer, collisions of ions with gas take place inside and near the ion trap, with the result that the high mass accuracy the time-flight-mass spectrometer originally has cannot be realized. In the known reference 3, mass spectrometry is carried out after ions accumulated in the ion trap have been transferred to the time-of-flight mass spectrometer representing a high vacuum chamber, so that MSn spectrometry can be performed at a high mass accuracy of less than 5 ppm the time-of-flight mass spectrometer has. Accordingly, the apparatus of reference 3 can be utilized sufficiently even for proteome analysis. However, the interior of the time-of-flight mass spectrometer needs to be maintained at high vacuum and hence, there are constraints imposed on the size of an inlet for introducing ions to the inside of the time-of-flight mass spectrometer. In addition, the inlet fills also the role of prescribing the width of an ion beam to realize high resolution and in this point, the size of the inlet is limited. On the other hand, spatial distribution of ions accumulated in the ion trap increases as the amount of ions to be accumulated increases. Ultimately, when the amount of accumulated ions exceeds a constant level, part of ions ejected out of the ion trap cannot pass through the inlet of the time-of-flight mass spectrometer. In other words, when the ion accumulation amount exceeds the constant level, signal intensity becomes saturated. Disadvantageously, the quantification accuracy becomes therefore poor. The proteome analysis aims at identifying protein with high accuracies and at the same time, examining the difference in the amounts of appearing protein. Accordingly, the quantification accuracy is important.
An object of the present invention is to provide an apparatus which can perform mass spectrometry and multi-stage MS/MS spectrometry with high mass accuracies and high quantitative accuracies.
According to the present invention, in an ion trap/time-of-flight mass spectrometer in which ions accumulated in an ion trap are introduced into a time-of-flight mass spectrometer, a field orthogonal to the traveling direction of ions is applied in the time-of-flight mass spectrometer to accelerate ions and time of flight required for the ions to reach a detector is measured, ions are accumulated for a constant period of time in the ion trap, accumulated ions are then ejected out of the ion trap and introduced into the time-of-flight mass spectrometer, total content of ions introduced into the time-of-flight mass spectrometer is measured, and an ion accumulation time for the next operation is determined on the basis of a result of measurement of the total ion content and a preset threshold value. The threshold value is an amount of ions or a value corresponding thereto when all ions or almost all the ions accumulated in the ion trap can pass through the inlet of the time-of-flight mass spectrometer.
It is desirable that the value of total content of ions reaching the interior of the time-of-flight mass spectrometer be measured accurately but disadvantageously, the accuracy will be impaired for the following reasons. Since time for ions to move from the ion trap to an orthogonal accelerator inside the time-of-flight mass spectrometer depends on mass-to-charge ratios of ions, only ions which are traveling through the inside of the accelerator at the time that the acceleration voltage is applied can undergo spectrometry. In other words, a mass range that can be subject to one operation of spectrometry (this is called a mass window) is limited. Accordingly, a measured amount of total ion content becomes inaccurate. In the present invention, this problem can be solved through three types of contrivance or features as below.
(1) A means for applying an auxiliary AC voltage to the ion trap is used to limit the mass range of ions that can be captured by the ion trap. The limited mass range is set within a range of mass window.
(2) A mass filter is interposed between the ion source and the ion trap and the pass band of the mass filter is set within the range of mass window.
(3) When measuring total content of ions that have passed through the inlet of the time-of-flight mass spectrometer, ions having passed though the slit are not accelerated orthogonally but are caused to travel rectilinearly so as to be detected by a detector.
Known reference 4 (C. Marinach, A. Brunot, C. Beaugrand, G. Bolbach, J.-C. Tabet, Proceedings of the 49th ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, Ill., May 27-31, 2001) discloses a method for forming a quasi-continuous beam by dispersing ions ejected out of an ion trap in the ejection direction. In this case, during passage of an ion beam through an accelerator, orthogonal acceleration of ions is conducted repeatedly. This method can eliminate the mass window but only ions which are traveling inside the accelerator can undergo spectrometry, raising a problem that ions pass through the accelerator during an interval of each spectrometry operation. Ions of lower m/z have higher speeds and therefore, the amount of ions passing through the accelerator depends on m/z. Accordingly, measurement of the value of total ion content still remains inaccurate. In this case, the means (3) as above is effective.